Use of Secretor, Lewis and Sialyl Antigen Levels in Clinical Samples as Predictors of Risk for Disease

ABSTRACT

An individual at risk for necrotizing enterocolitis and related disorders can be identified by measuring the level of at least one secretor antigen in a biological sample from the individual and comparing the measured level of the at least one secretor antigen to a predetermined value or a predetermined range of values. Among the secretor antigens which can be measured are: the H-1, H-2, Lewis b  and Lewis y  antigens and derivatives thereof (e.g., a sialylated form of Lewis a, Lewis x, Lewis b, Lewis y; H-1, H-2, Lewis a, Lewis x, Lewis b or Lewis y).

RELATED APPLICATION INFORMATION

This application is a divisional application of U.S. patent applicationSer. No. 13/006,795, filed Jan. 14, 2011, which is a continuationapplication of U.S. patent application Ser. No. 12/205,089, filed Sep.5, 2008, which claims the benefit of U.S. provisional application Ser.No. 60/970,902, filed Sep. 7, 2007. The contents of the priorapplications are incorporated herein by their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under HD013021 awardedby the National Institutes of Health. The government has certain rightsin this invention.

TECHNICAL FIELD

This disclosure relates to the field of medical diagnostics and moreparticularly to materials and methods for assessing and treatinginflammatory and infectious disorders in infancy.

BACKGROUND

Inflammatory and infectious disorders in infancy can be associated withserious morbidity and mortality. Inflammatory and infectious disordersthat occur perinatally, for example, necrotizing enterocolitis (NEC),sepsis, and chorioamnionitis are major contributors to neonatalmortality rates. NEC, the most common gastrointestinal medical and/orsurgical emergency occurring in neonates, occurs in 7-13% of very lowbirthweight infants, and is characterized by bowel injury and intestinalnecrosis. NEC mortality rates overall are in the range of 20-40%, andmortality rates for premature infants have been reported to exceed 50%.Surgical intervention is required in about 30% of cases, andsurgery-associated mortality is reported to be as high as 50%.

Survivors of inflammatory and infectious disorders of infancy can havesignificant short-term and long-term morbidities, including metaboliccomplications, recurrent infections, poor neurodevelopmental outcomes,and poor growth outcomes, that in some instances may require repeatedsurgical intervention and prolonged hospitalization.

SUMMARY

The methods described herein are based, in part, on our discovery ofmethods for assessing whether an infant is at high risk of death, orlikely to develop certain inflammatory or infectious disorders, forexample, necrotizing enterocolitis, sepsis, gastrointestinal infections,respiratory infections, and urinary tract infections. Disclosed hereinis a method of identifying an individual at risk for necrotizingenterocolitis, the method comprising:

(a) measuring the level of at least one secretor antigen in a biologicalsample from the individual, and

(b) comparing the measured level of the at least one secretor antigen toa predetermined value or a predetermined range of values,

wherein the individual is at risk for necrotizing enterocolitis if themeasured level of the at least one secretor antigen differs from thepredetermined value or is outside the predetermined range of values.

In various embodiments: the individual is an infant (e.g., infant is aneonate, a low birthweight infant, an extremely low birthweight infantor a premature infant); the at least one secretor antigen comprises anα1,2-linked fucose antigen and/or an α2,3 sialylated antigen; the atleast one secretor antigen is selected from the group consisting of: theH-1, H-2, Lewis^(b) and Lewis^(y) antigens and derivatives thereof; thederivative is a sialylated form of Lewis a, Lewis x, Lewis b or Lewis y;the derivative is a sulfated form of H-1, H-2, Lewis a, Lewis x, Lewis bor Lewis y; the biological sample is a bodily fluid or a tissue; thebodily fluid comprises saliva, blood, plasma, serum, urine, stool,amniotic fluid, hmucus, tears or lymph; the bodily fluid comprisessaliva; the measuring step comprises an immunoassay; the at least onesecretor antigen is selected from the group consisting of: H-1, H-2,Lewis^(b) and Lewis^(y) and derivatives thereof and wherein theindividual is determined to be risk for necrotizing enterocolitis if themeasured level is below the predetermined value or below thepredetermined range of values; the predetermined value or thepredetermined range of values represents the average level of the atleast one secretor antigen in a population of individuals determined tobe secretors; the individual is determined to be at risk for necrotizingenterocolitis when the measured level of at least one secretor antigenis at least 10% less than the average level found in a controlpopulation of secretors; the at least one secretor antigen is the H-2antigen.

In some cases the at least one antigen is sialyl Lewis a or derivativethereof and the individual is determined to be risk for necrotizingenterocolitis if the measured level is above the predetermined value orabove the predetermined range of values. In some embodiments of thismethod: the infant is a neonate, a low birthweight infant, an extremelylow birthweight infant or a premature infant, the biological sample is abodily fluid or a tissue; the bodily fluid comprises saliva, blood,plasma, serum, urine, stool, amniotic fluid, mucus, tears or lymph; thebodily fluid comprises saliva; the measuring step comprises animmunoassay; and the predetermined value or the predetermined range ofvalues represents the average level of the at least one secretor antigenin a population of individuals determined to be secretors.

Also disclosed is a method of identifying an individual at risk fordeveloping a gastrointestinal disorder, the method comprising:

(a) measuring the level of at least one secretor antigen in a biologicalsample from the individual, and

(b) comparing the measured level of the at least one secretor antigen toa predetermined value or a predetermined range of values,

wherein the individual is at risk for a developing a gastrointestinaldisorder if the measured level of the at least one secretor antigendiffers from the predetermined value or is outside the predeterminedrange of values.

In various embodiments of this method: the individual is an infant(e.g., a neonate, a low birthweight infant, an extremely low birthweightinfant or a premature infant); the measured antigens comprise anα1,2-linked fucose antigen and/or an α2,3 sialylated antigen; thesecretor antigen is selected from the group consisting of: the H-1, H-2,Lewis^(b) and Lewis^(y) antigens and derivatives thereof; the derivativeis a sialylated form of Lewis a, Lewis x, Lewis b or Lewis y; thederivative is a sulfated form of Lewis a, Lewis x, Lewis b or Lewis y;the biological sample is a bodily fluid or a tissue; the bodily fluidcomprises saliva, blood, plasma, serum, urine, stool, amniotic fluid,mucus, tears or lymph; the measuring step comprises an immunoassay; theat least one secretor antigen is selected from the group consisting of:H-1, H-2, Lewis^(b) and Lewis^(y) and derivatives thereof and whereinthe individual is determined to be risk for developing agastrointestinal disorder if the measured level is below thepredetermined value or below the predetermined range of values; thepredetermined value or the predetermined range of values represents theaverage level of the at least one secretor antigen in a population ofindividuals determined to be secretors; the individual is determined tobe at risk for necrotizing enterocolitis when the measured level of theat least one secretor antigen is at least 10% less than average levelfound in control population of secretors; the at least one antigen isthe H-2 antigen; the gastrointestinal disorder is gastrointestinalinflammation; the gastrointestinal disorder is gastrointestinalinfection; the disorder is late onset sepsis; an the gastrointestinalinfection comprises infection with one or more of Staphylococcus spp.,Staphylococcus aureus, Escherichia coli, Streptococcus spp.,Enterobacter spp., Klebsiella spp., Bacillus spp., Serratia spp.,Candida spp, Norwalk and other Noroviruses, Campylobacter spp, Vibriocholerae, Bacteriodes spp., Clostridiae, Giardia.

Also disclosed is a method comprising:

(a) measuring the level of at least one secretor antigen in a biologicalsample from the individual, and

(b) comparing the measured level of the at least one secretor antigen toa predetermined value or a predetermined range of values,

(c) determining that the individual is at risk for necrotizingenterocolitis if the measured level of the at least one secretor antigendiffers from the predetermined value or is outside the predeterminedrange of values; and

(d) taking steps to treat or reduce the risk for necrotizingenterocolitis if the individual is determined to be at risk fornecrotizing enterocolitis.

In this method (d) can comprise administering to the individual one ormore of α1,2 fucosyl glycans, probiotic organisms or prebiotics.

Also disclosed is a method of identifying an individual at risk fornecrotizing enterocolitis, the method comprising:

(a) measuring the level of FUT2 protein or mRNA encoding FUT2 in abiological sample from the patient;

(b) comparing the measured level of level of FUT2 protein or mRNAencoding FUT2 to a predetermined value or a predetermined range ofvalues,

wherein the individual is at risk for necrotizing enterocolitis if themeasured level of FUT2 protein or mRNA encoding FUT2 is below apredetermined value or a predetermined range of values.

Disclosed herein is a method of identifying an individual at risk fornecrotizing enterocolitis, the method comprising:

(a) providing a biological sample from the individual;

(b) determining whether the individual harbors a FUT2 gene havinggenetic change that reduces the expression or activity of FUT2,

wherein the individual is at risk for necrotizing enterocolitis if theindividual harbors a FUT2 gene having genetic change that reduces theexpression or activity of FUT2.

Disclosed herein is a method of identifying an individual (e.g., aninfant) at risk for death, the method comprising:

(a) measuring the level of at least one secretor antigen in a biologicalsample from the individual, and

(b) comparing the measured level of the at least one secretor antigen toa predetermined value or a predetermined range of values,

wherein the individual is at risk for necrotizing enterocolitis if themeasured level of the at least one secretor antigen differs from thepredetermined value or is outside the predetermined range of values.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1: Fucosyltransferase and Fut2 mRNA expression in mouse colon. (A)α1,2/3-fucosyltransferase in conventional [CONV], germ-free (GF) andex-germ-free (XGF) mice; (B) α1,2/3-fucosyltransferase activity and (C)Fut2 mRNA in the CONV, bacteria-depleted (BD) and bacteria-repleted(XBD) mice; (D) α1,2-fucosylated glycan expression detected by UEA-1lectin and corresponding Nomarski image.

FIG. 2: (A) TLR4 expression is necessary for bacterial activation ofintestinal fucosylation. (B) TLR4 ligands are sufficient for activationof mucosal fucosylation in bacteria-depleted mice.

FIG. 3: Monocolonization by B. fragilis induces intestinal fucosylation.

FIG. 4: Histograms indicate the distribution of H-2, Le y, and sLe aantigens measured in saliva as optical density (O.D.) values comparing24 cases of NEC or death (top row) and 168 controls (bottom row).Samples were collected at 8-14 days (week 2). The table indicates theincidence of NEC or death, the relative risk and p-value comparing therisk groups identified by CART analysis. The triangle symbol indicatesthe cutpoint in continuous values of each antigen identified by CARTanalysis to optimally distinguish between a high and low risk group(node). This cutpoint is applied in the table for each antigen. For H-2only: the O.D. value distribution differs significantly (P=0.004,Wilcoxon Mann Whitney test) comparing NEC and death cases with controls.

FIG. 5: Schematic depiction of CART analysis.

FIG. 6: Scatterplot of H-2 and sLe A antigen optical density (O.D.)values by EIA measured from a saliva sample collected in week 2 (days8-14) from 192 ELBW (<1000 grams) infants in Cincinnati NICUs; 24(12.5%) developed NEC or died. The high and low risk sets weresystematically identified by CART analysis to minimize misclassificationof cases and non-cases. The H-2 cut-point for high risk was identifiedas an O.D. <0.627 (lowest 38% of values, including non-secretorinfants). In the H-2 high risk group, 18 cases occurred in 73 infants(incidence=24.7%) compared to 6 cases in 119 infants (incidence=5.0%;relative risk [RR]=4.9, 95% confidence interval [CI]=2.0 to 11.8,P<0.0001). The sLe^(a) cutpoint for high risk was identified as anO.D. >0.318 (highest 76% of values). The infants in both high riskgroups (defined by low H-2 and high sLe^(a) O.D. values) creates thehigh risk set indicated in the figure of 17 cases in 54 infants(incidence=31.5%) compared to all other infants combined, who define thelow risk set, which comprises 7 cases in 138 infants (incidence=5.1%).This combined classification resulted in a highly significant split ofhigh vs. low risk (RR=6.2, 95% CI 2.7 to 14.1, P<0.0001).

FIG. 7: Sequence of human “secretor” FUT2 mRNA.

FIG. 8: Sequence of human “secretor” FUT2 polypeptide.

DETAILED DESCRIPTION

The ABH and Lewis histo-blood group antigens are carbohydratesrepresenting the terminal structures of glycan chains. The H-typehisto-blood group antigens, for example, the H type 1 and 2, theLewis^(b) and the Lewis^(y) antigens, are characterized by a fucoseterminus in an α1,2 linkage. In mammals, the H-type histo-blood groupantigens are found on a wide range of tissue types including epithelialcells of organs that are in direct contact with the externalenvironment, e.g., the upper respiratory tract, the nasal epithelium andtrachea, as well as the genito-urinary tract, ureter and vagina, as wellas on erythrocytes, some neurons of the peripheral nervous system,thymus epithelium and the skin. In several human populations, about 80%of individuals also express H-type histo-blood group antigens in asoluble form in biological fluids, for example, saliva, breast milk,serum, tears, sweat and semen. In about the remaining 20% of individualsin these human populations, the soluble H-type histo-blood groupantigens are either absent from, or found at extremely low levels, inbiological fluids. These two phenotypes have been designated as“secretors” and “non-secretors” respectively and the soluble H-typehisto-blood group antigens are typically referred to as secretorantigens. In other human populations the percentage of individuals whoare non-secretors is lower than 20%.

The basis for the phenotypic difference between the secretor andnon-secretor subpopulations stems from genetic polymorphisms in the FUT2gene encoding the enzyme fucosyl transferase 2 also referred to in theart as alpha(1,2)fucosyltransferase 2, EC 2.4.1.69, SE 2, SEC2,Fucosyltransferase-2 (secretor), GDP-L-fucose:beta-D-galactoside2-alpha-L-fucosyltransferase 2, Galactoside 2-alpha-L-fucosyltransferase2, Secretor blood group alpha-2-fucosyltransferase, Secretor factor, andtransmembrane protein 2. The FUT2 gene is also referred to in the art asthe secretor gene (Se). The FUT2 gene product, FUT2, catalyzes theformation of an α-L-fucosyl-1,2-β-D-galactosyl-R structure fromGDP-β-L-fucose and β-D-galactosyl-R, where R can be a glycoprotein or aglycolipid. FUT2 is expressed in many organs that generally are ofendodermal origin including gut, pharynx, liver, respiratory tract,bladder, urethra and endocrine glands, although within those organs FUT2expression is also a function of the differentiation pattern of thetissue, e.g., keratinized vs. non-keratinized squamous epithelium, ductsvs. acini of glandular tissues, as well as the particular cell type,e.g, secretory vs. ciliated cells in the endometrium, and mucous vs.serous cells in the salivary gland. Secretors express functional FUT2;non-secretors fail to express functional FUT2. Accordingly, solubleH-type histo-blood group antigens are not synthesized and therefore notsecreted into biological fluids of non-secretors.

The experiments described in the Examples indicate that the secretorstatus and the expression of sialyl glycan epitopes of an infant arecorrelated with risk of NEC and death. More specifically, extremely lowbirthweight infants (ELBW) or premature infants who expressed little orno H-2 antigens in saliva, as well as infants who expressed elevatedlevels of sialyl Lewis a antigens in saliva, are significantly morelikely to experience adverse clinical outcomes such as NEC, late onsetsepsis and death compared to infants who express moderate or high levelsof H-2 antigen or infants who express little or no sialyl Lewis aantigens in saliva.

Disclosed herein are materials and methods relating to theidentification of individuals as risk of developing an inflammatory orinfectious disorder, for example, NEC, gastrointestinal infections, orlate onset sepsis. More specifically, an infant can be identified asbeing at risk for an inflammatory or infectious disorder, for example,NEC, gastrointestinal infection or late onset sepsis, by assessing thesecretor status of the infant. In some embodiments, an infant who doesnot express or who expresses low levels of secretor antigens relative toa reference sample can be classified as being at risk for NEC,gastrointestinal infection or late onset sepsis. In other embodiments,an infant who expresses elevated levels of sialyl Lewis a (sLe^(a))antigen relative to a reference sample can be classified as being atrisk for NEC, gastrointestinal infection or late onset sepsis.

Also provided herein are methods of treatment and management ofindividuals at risk of developing an inflammatory or infectiousdisorder, for example, NEC, gastrointestinal infections, or late onsetsepsis. In some embodiments, an individual identified by secretor statusas being at risk of developing NEC, gastrointestinal infection or lateonset sepsis can be treated with specific therapies that includeprotective agents, e.g., probiotic organisms or prebiotic agents,including α1,2 fucosyl glycans. In some embodiments, the course oftreatment of an individual identified by secretor status as being atrisk of developing NEC, gastrointestinal infection or late onset sepsiscan be evaluated by assessing the level of secretor antigens in themother's milk being provided to her infant and, based on the levels ofsecretor antigens in the food source relative to a reference sample,administering to the infant specific therapies that include protectiveagents, e.g., α1,2 fucosyl glycans, probiotic organisms or prebiotics.

Secretor Antigens

Secretor antigens, i.e., the H type 1 and 2, the Lewis^(b) and theLewis^(y) antigens, are glycans that include a fucose terminus in anα1,2 linkage. The term glycans as used herein refers to a compound oftwo or more subunit monosaccharide units joined together by a glycosidicbond, i.e., a covalent bond between an anomeric hydroxyl group, in α orβ configuration, of one monosaccharide and any available hydroxyl groupin a second monosaccharide, regardless of additional modifications e.g.,linkages to other additional monosaccharide units, polypeptides, lipidsor other biological or nonbiological molecules. We may refer to asaccharide polymer containing a small number, typically three to 35 ormore component sugars as an oligosaccharide. All oligosaccharidesdescribed herein are described with the name or abbreviation for thenon-reducing saccharide (i.e., Gal), followed by the configuration ofthe glycosidic bond (α or β), the ring bond (1 or 2), and then the nameor abbreviation of the next saccharide (i.e., GlcNAc) toward thereducing end of the molecule. For a review of standard glycobiologynomenclature see, Essentials of Glycobiology, Varki et al., eds., 1999,Cold Spring Harbor Laboratory Press.

The specific form of the secretor antigen can vary and depends, in part,upon the structure of the minimal disaccharide precursor, or coresequence, from which the particular antigen was assembled. The coresequence can be either the lacto type I structure, galactose (β1-3)N-acetylglucosamine-R, which we abbreviate here as {Gal (β1-3)GlcNAc}-Ror the lacto type II structure galactose (β1-4) N-acetylglucosamine-R,which we abbreviate here as {Gal(β1-4)GlcNAc}-R. In the minimal,unconjugated core sequence, R can be H or other small molecule radicals.The disaccharide precursor can also be conjugated to longer glycans asoligosaccharides, or as the glycan moieties of glycolipids, peptides,proteins, mucins, or other macromolecules.

Thus, for example, the H-1 antigen is derived from the type I precursorby FUT2 (or FUT1) catalyzed addition of a fucose residue to thegalactose moiety of the type I precursor in an α1,2 linkage to generatethe structure {Fucose(α1-2) Galactose (β1-3) N-acetylglucosamine, whichwe abbreviate as {Fuc (α1-2) Gal (β1-3) GlcNAc}. The H-1 antigen is astructural precursor to another secretor antigen, Lewis^(b). TheLewis^(b) secretor antigen includes the H-1 structure plus a secondfucose residue in a non-terminal α1,4 linkage to the GlcNAc moiety inthe configuration {Fucose(α1-2) Galactose (β1-3)[Fucose(α1-4)]N-acetylglucosamine} which we abbreviate as {Fuc (α1-2)Gal (β1-3) [Fuc (α1-4)] GlcNAc}.

Correspondingly, the H-2 antigen is derived from the type II precursorby FUT2 (or FUT1) catalyzed addition of a fucose residue to thegalactose moiety of the type II precursor in an α1,2 linkage to generatethe structure {Fucose(α1-2) Galactose (β1-4) N-acetylglucosamine}, whichwe abbreviate as {Fuc (α1-2) Gal (β1-4) GlcNAc}. The H-2 antigen is astructural precursor to another secretor antigen, Lewis^(y). TheLewis^(y) secretor antigen includes the H-2 structure plus a secondfucose residue in a non-terminal α1,3 linkage to the GlcNAc moiety inthe configuration {Fucose(α1-2) Galactose (β1-4)[Fucose(α1-3)]N-acetylglucosamine} which we abbreviate as{Fuc (α1-2) Gal(β1-4) [Fuc (α1-3)] GlcNAc}.

A secretor antigen may comprise a single α1,2 fucose substituted coresequence, wherein R is H or other small radicals. Alternatively, asecretor antigen can comprise a repetitive series of substituted coresequences, wherein R is another core sequence. The single core sequenceor a repetitive core sequence may be present within a larger sugar.Accordingly, a secretor antigen-containing oligosaccharide can be, forexample, a trisaccharide, a tetrasaccharide, a pentasaccharide, and soon. A secretor antigen can also be covalently linked to anothermacromolecule, e.g., a polypeptide or a lipid. The single substitutedcore sequence can also be linked directly to a polypeptide or lipid,e.g., R can be a protein or a lipid or can be present in apolysaccharide that is bound to a polypeptide or lipid. Secretorantigens may be covalently linked to polypeptides via N-linkedglycosylation, that is, through an asparagine residue, or via O-linkedglycosylation, for example, through serine, threonine, hydroxylproline,tyrosine or other hydroxyl containing residue. Glycoproteins thatinclude secretor antigens include, for example without limitation,mucins, bile-salt-stimulated lipase (BSSL), and lactadherin. Examples ofsecretor antigen-bearing lipids include, without limitation, H-1glycolipid, H-2 glycolipid, Le^(x) glycolipid, and Le^(y) glycolipid.

Lewis Antigens

The Lewis antigens can be present as any glycan containing Lewisepitopes, that is, containing H-1, H-2, Le^(a), Le^(b), Le^(x), and/orLe^(y) epitopes, which are α1,3 or a1,4-linked fucosylatedoligosaccharide moieties. This includes free oligosaccharides,glycolipids, glycoproteins, mucins, glycosaminoglycans, andglycopeptides. Individuals can express glycans that express the H-1,H-2, Le^(a), Le^(b), Le^(x), and/or Le^(y) epitopes.

Sialyl Antigens

The sialyl antigens can be present as any glycan containing sialylatedantigens, including free oligosaccharides, glycolipids (e.g.,gangliosides), glycoproteins, mucins, glycosaminoglycans, andglycopeptides. Individuals can express sialylated epitopes, includinggangliosides and other glycans that express the sialyl Lewis a(sLe^(a)), sLe^(b), sLe^(x), and/or sLe^(y) epitopes.

Assaying Secretor Antigens

The level of one or more secretor antigens can be measured in anybiological fluid known in the art to comprise secretor antigens.Examples of biological fluids include, without limitation, saliva,serum, blood plasma, breast milk, amniotic fluid, sweat, urine, tears,mucus, lymph, and stool. Biological fluid samples can be collected froman individual using any standard method known in the art that results inthe preservation of secretor antigen structure. Saliva samples may becollected using cotton swabs, wipes, suction, scraping, or by having theindividual rinse the mouth and expectorate into a tube or collector.Blood samples can be obtained via venous puncture techniques. Serumsamples can be prepared from whole blood using standard methods such ascentrifuging blood samples that have been allowed to clot. Plasmasamples can be obtained by centrifuging blood samples that were treatedwith an anti-coagulant such as heparin. Breast milk can be collected bymanual or mechanical expression. Biological fluid samples can be assayedfor secretor antigens immediately following collection. Alternatively,or in addition, a biological fluid sample can be stored for lateranalysis using methods known in the art that preserve secretor antigenstructure, e.g., freezing, drying, or freeze drying.

After determining the levels of specific secretor antigens in abiological sample, these levels can be compared with those of standardreference levels. Standard reference levels typically represent theaverage secretor antigen levels derived from a large population ofindividuals. The reference population may include individuals of similarage, body size, ethnic background or general health as the individual inquestion. The FUT2 genotype of the reference population may or may notbe known. Thus, the secretor antigen levels in a patient's sample can becompared to values derived from: 1) individuals who express wild-typeFUT2 and whose bodily fluids contain secretor antigens; 2) individualswho express variant forms of FUT2 and have moderate to low FUT2 activityand whose bodily fluids contain low levels of secretor antigens; or 3)individuals who have little or no FUT2 activity and whose bodily fluidslack secretor antigens.

In general, an elevated level of secretor antigen can be any level of asecretor antigen that is greater than either the level of a secretorantigen found in a control sample or greater than the average level of asecretor antigen found in samples from a population of normal healthyindividuals who are secretors. A reduced level of a secretor antigen canbe any level of a secretor antigen that is less than either the level ofa secretor antigen found in a control sample or less than the averagelevel of a secretor antigen found in samples from a population of normalhealthy individuals who are secretors. Any population size can be usedto determine the average level of a secretor antigen found in samplesfrom a population of normal healthy individuals that are secretors. Forexample, a population of between 2 and 250, e.g., 2, 3, 4, 5, 10, 15,20, 25, 30, 40, 50, 100, 150, 200, 250 or more individuals can be usedto determine the average level of a secretor antigen in samples from apopulation of normal healthy individuals, with greater accuracy in themeasurement coming from larger sample populations.

A reduced level of a secretor antigen can be 10, 20, 30, 50, 60, 70, 80,90, 100, percent lower than that level found in a control sample orlower than the average level of a secretor antigen found in samples froma population of normal healthy individuals. In some cases, a reducedlevel of a secretor antigen can be 2, 3, 4, 5, 10, 20, 50 or more foldlower than that level found in a control sample or the average level ofa secretor antigen found in samples from a population of normal healthyindividuals.

In some cases, a reference chart can be used to determine whether or nota particular level of a specific secretor antigen in a sample is low ornormal relative to a control sample or a larger population. For example,a reference chart can contain the normal range of secretor antigenlevels found in healthy infants of the same age, gestational age, ethnicbackground or general health as the individual in question. Using thisreference chart, any level of a secretor antigen measured in a samplecan be classified as being low, normal, or elevated relative to acontrol sample or relative to an average value derived from a largerpopulation.

Alternatively, or in addition, the level of a secretor antigen in abiological sample can be “normalized” against the level of one or moreadditional biological markers, for example another histo-blood groupantigen, such as a P or sialyl antigen, whose expression is independentof the secretor status of the individual. That is, the levels of theadditional marker can be evaluated in parallel with those of thesecretor antigen, either at the same time or on a separate occasion. Theadditional marker can serve as an internal control for samplepreparation, handling and storage as well as day-to-day assayvariability. The values for the level of a secretor antigen and theadditional marker may be expressed as a ratio and the ratio may becompared to similar ratio obtained for a reference sample or population.Examples of useful second markers include, but are not limited to, Lewisantigens whose expression is independent of the secretor (FUT2)expression, i.e., Lewis a and Lewis x. A Lewis antigen generallyincludes carbohydrates having as a core sequence either the lacto type Istructure or the lacto type II structure substituted with one or morefucosyl residues. Thus, for example, a useful second marker can be Lewisa {Galactose (β1-3)[Fucose (α1-4)]N-acetylglucosamine}, which weabbreviate as {Gal (β1-3) Fuc (α1-4) GlcNac}, or Lewis×{Galactose(β1-4)[Fucose (α1-3)]N-acetylglucosamine, which we abbreviate as {Gal(β1-4) [Fuc (α1-3)] GlcNAc}, or the sialylated, sulfated, orsulfo-sialylated forms of these epitopes. Other commonly expressednon-Lewis blood group antigens could also be used, such as Lua (Lutherana), P1 & P2 (the major antigens of the P blood group system), M&N, Fya&Fy b (antigens of the Duffy system), etc.

In addition, since stochastic variations in individual gene expressionlevels are common in biological systems, it may be desirable tonormalize the level of a secretor antigen against a panel of two or moreadditional biological markers whose expression is known to beindependent of the secretor status of the individual. This strategy maylead to greater accuracy in determining secretor antigen levels.

Once the relative level of a secretor antigen in an individual relativeto that of a reference sample has been calculated, the individual'srelative risk for gastroenteritis, necrotizing enterocolitis, late onsetsepsis or death can be assessed. Any statistical method known in the artfor evaluating relative risk may be used. One suitable method isClassification and Regression Tree (CART) characteristic curve analysis.CART analysis belongs to a family of nonparametric regression methodsand is based on recursive partitioning to build a decision tree thatoptimizes the classification of individuals into high and low-riskgroups. It can be applied to systematically identify cutpoints incontinuous variables that maximize predictive value and minimizemisclassification of cases and non-cases based on a balance between thesensitivity (i.e., the number of true cases detected) and thespecificity (i.e., the accuracy) of a test. These two variables may alsobe considered positive predictive value and negative predictive value,and are correlated with diagnostic accuracy. The decision tree producedby CART analysis can be validated through Receiver Operating Curve (ROC)analysis to determine the area under the curve (AUC), which indicatesthe effectiveness of the relationship of the decision tree todiscriminate between cases and non-cases.

In one example, CART analysis indicates that an individual classified asa low or non secretor, i.e, in the 38th percentile or below of H-2salivary expression (as measured by O.D. values), has an increased riskfor NEC and death. Thus, an individual whose levels of secretor antigensare in the 38th, 33rd, 30th, 25th, 20th, 15th, 10th, 5th, or lowerpercentile is 4 to 5 times more likely to suffer from NEC or death thanindividuals whose secretor antigen levels are above the 38th percentile.Among those who express little or no secretor antigen, the highest riskgroup is defined by those who also express high sialyl Lewis a antigen.Infants who express little or no secretor antigen and high sialyl Lewisa antigen have more than 6-fold the risk of NEC or death relative to allother infants. Thus, the high risk group can be defined either by littleor no H-2 antigen alone or by the combination of little or no H-2 andmoderate to high sialyl Lewis a antigen. Based on this comparison, aswell as on other clinical indices, a clinician can predict thelikelihood that a patient is at risk for NEC, and adjust treatmentregimens accordingly.

The secretor status of an individual can be determined in a variety ofways. The level of a secretor antigen can be measured directly in abiological fluid. For example, the level of a secretor antigen can bemeasured using immuno-based assays, e.g., ELISA assays,radioimmunoassays, one or two dimensional gel electrophoresis coupledwith immunodetection, or by using surface plasmon resonance-basedbiosensors, or by using chromatographic techniques, e.g., highperformance liquid chromatography (HPLC) or gas chromatography (GC), orby spectrometry, e.g., mass spectrometry. Alternatively, or in addition,FUT2 activity can be measured in a biological sample using standardenzymology methods. In addition, FUT2 mRNA levels may be quantified (asa surrogate for levels of FUT2 activity in a biological sample) using avariety of methods well known to the art, e.g. RT-PCR or quantitativeRT-PCR (for example: Kroupis C, Stathopoulou A, Zygalaki E, Ferekidou L,Talieri M, Lianidou E S. Clin Biochem. 2005 January; 38(1):50-7, Dyer J,Chisenhall D M, Mores C N. J Virol Methods. 2007 October; 145(1):9-13),or quantitative hybridization-based techniques such as cDNA oroligonucleotide microarrays (for example: Duggan D. J., Bittner M., ChenY., Meltzer P. and Trent J. M. Nat Genet. 21(1 Suppl):10⁻⁴ (1999);Cheung V. G., Morley M., Aguilar F., Massimi A., Kucherlapati R. andChilds G. Nat Genet. 21(1 Suppl):15-9 (1999)).

The human secretor FUT2 mRNA sequence is shown in FIG. 7 (GenBank®Accession No NM_(—)000511.4) and the human secretor FUT2 proteinsequence in shown in FIG. 8 (GenBank® Accession No. NP 000502.4).

Finally, the FUT2 genotype of an individual can be determined by singlenucleotide polymorphism analysis (SNP) or RT-PCR-based techniques.

Immunoassays

Immunoassay methods are well known to those in the art. Antibodyreagents that detect specific secretor Lewis antigens, e.g., H-1, H-2,Lewis^(b), and Lewis^(y), and other Lewis antigens. e.g., Lewis^(a) andLewis^(x), can be generated using standard methods for antibodyproduction or purchased from commercial sources. Antibodies can bemonoclonal or polyclonal or any combination thereof. Useful antibodiescan include: monoclonal and polyclonal antibodies, single chainantibodies, chimeric antibodies, bifunctional/bispecific antibodies,humanized antibodies, human antibodies, and complementary determiningregion (CDR)-grafted antibodies, that are specific for the secretor orglycan epitope, and also include antibody fragments, including Fab,Fab′, F(ab′)2, scFv, Fv, camelbodies, or microantibodies. In additionnucleic acid or peptide aptamer reagents capable of specifically bindingto and detecting specific secretor antigens can be generated usingstandard published methods (e.g. The use of aptamers in large arrays formolecular diagnostics, Brody E. N., Willis M. C., Smith J. D., JayasenaS., Zichi D. and Gold L. Mol Diagn 4(4):381-8 (1999)).

Thus, in some embodiments, a specific anti-H-1, anti-H-2,anti-Lewis^(b), or anti-Lewis^(y) monoclonal antibody or aptamer canhave a binding affinity less than about 1×10⁵ Ka for an antigen otherthan H-1 and H-2, Lewis^(b), and Lewis^(y), respectively. In someembodiments, the anti-H-1, anti-H-2, anti-Lewis^(b), or anti-Lewis^(y)antibody is a monoclonal antibody that binds to H-1, H-2, Lewis^(b), andLewis^(y) with an affinity of at least 1×10⁸ Ka.

Any form of H-1 and H-2, Lewis^(b), and Lewis^(y) can be used togenerate the anti-H-1 anti-H-2, anti-Lewis^(b), and anti-Lewis^(y)antibodies or aptamers respectively, including, minimal trisaccharide ordisaccharide structures or epitope-bearing fragments thereof, and anyglycan containing these epitopes. Highly suitable anti-H-1 anti-H-2,anti-Lewis^(b), and anti-Lewis^(y) antibodies or aptamers are those ofsufficient affinity and specificity to recognize and bind to theirrespective targets in vivo. As used herein, the term epitope refers toan antigenic determinant of a glycan.

Specific carbohydrate binding antibodies or aptamers can be moleculesthat 1) exhibit a threshold level of binding activity; and/or 2) do notsignificantly cross-react with known related glycan molecules. Thebinding affinity of an antibody or aptamer can be readily determined byone of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949). In some embodimentsthe antibodies or aptamers can bind to their target epitopes or mimeticdecoys with at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold,10³-fold, 10⁴-fold, 10⁵-fold, 10⁶-fold or greater affinity for thetarget glycan than to other glycans having some homology to the targetglycan.

In some embodiments the antibodies or aptamers bind with high affinityto H-1 or H-2, Lewis^(b), or Lewis^(y) of 10⁴M or less, 10⁻⁷M or less,10⁻⁹M or less or with subnanomolar affinity (0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, 0.1 nM or even less). In some embodiments the bindingaffinity of the antibodies or aptamers for H-1 or H-2, Lewis^(b), orLewis^(y) is at least 1×10⁶ Ka. In some embodiments the binding affinityof the antibodies or aptamers for H-1 or H-2, Lewis^(b), or Lewis^(y) isat least 5×10⁶ Ka, at least 1×10⁷ Ka, at least 2×10⁷ Ka, at least 1×10⁸Ka, or greater. Antibodies or aptamers may also be described orspecified in terms of their binding affinity to H-1 and/or H-2,Lewis^(a), Lewis^(b), Lewis', and/or Lewis^(y). In some embodimentsbinding affinities include those with a K_(d) less than 5×10⁻² M, 5×10⁻⁵M to 5×10⁻⁷ M, 5×10⁻⁸ M to 5×10⁻¹° M, 5×10⁻¹² M to 5×10⁻¹⁴ M, or less.

Antibodies or aptamers can be purified by chromatographic methods knownto those of skill in the art, including ion exchange and gel filtrationchromatography (for example, Caine et al., Protein Expr. Purif. (1996)8(2):159-166). Alternatively or in addition, antibodies or aptamers canbe purchased from commercial sources, for example, Abcam, BiovendorLaboratory, Calbiochem, Signet Laboratories, Accurate Chemical andScientific Corporation, and EMD.

Levels of secretor antigens can be measured in a biological sample usingany immunoassay format known to those of skill in the art. For example,in non-competitive immunoassays, the secretor antigen is “sandwiched”between two antibodies, a capture antibody and a detection antibody.Typically, the capture antibody is bound either covalently ornon-covalently to a solid phase, such as a tube or well, and thedetection antibody is conjugated to an enzyme in the case of ELISAassays, or is radiolabeled in the case of RIA assays. In ELISA assays,the detection antibody can be covalently linked to an enzyme, or canitself be detected by a secondary antibody that is linked to an enzymethrough bioconjugation. Specific antibody binding is analyzed by addingan enzymatic substrate, e.g., a chromogenic or fluorogenic molecule,which produces a detectable quantifiable signal upon cleavage. Specificantibody binding in RIA assays is determined by measuring the levels ofradioactivity bound to the support.

In a competitive immunoassay, the antigen (analyte) in the samplecompetes with the labeled antigen (tracer) for a limited number ofantibody binding sites. The bound antigen is separated from the excessanalyte not bound to the antibody. The amount of the analyte in theunknown sample is inversely proportional to the amount of labeledantigen, as measured in a gamma counter or spectrophotometer. Examplesof competitive immunoassays include double antibody radioimmunoassays(RIAs), coated tube RIAs and coated well enzyme immunoassays (EIAs).Some examples of solid supports that can be used include plates, tubes,polystyrene beads, nylon, nitrocellulose, cellulose acetate, glassfibers and other types of porous polymers. Suitable labels includeradionuclides, fluorophores, chemiluminescent labels, bioluminescentlabels, enzymes, for example, as used in ELISA systems, dyes orparticles such as colloidal gold or quantum dots.

Assay systems and kits designed to detect one or more specific secretorand Lewis antigens simultaneously are also within the scope of themethod. The kits may be dip-stick, flow-through or migratory in designas well as other formats known to those skilled in the art. If desired,the assays can be automated to insure standardization and obtain higherthroughput.

FUT2 Activity Assays

FUT2 activity can be measured in a biological sample using any standardmethod known in the art that is specific for FUT2. For example, afucosyltransferase assay can be performed in a 20 μl reaction volumecontaining 3 μM GDP-[¹⁴C]fucose, 5 mM ATP, 25 mM sodium phosphate, pH6.0, 40 μg of total protein from cell extracts and phenylβ-D-galactoside or asialofetuin as acceptor substrates. Reactionmixtures are incubated at 37° C. for 2 h, and terminated by the additionof 1 ml of water. The hydrophobic fucosylated phenyl β-D-galactosideproducts are purified by passing the reaction products through a C₁₈reverse-phase column. Radiolabelled asialofetuin products are purifiedby filtration through microfiber membranes (GF/C; Whatman) andradioactivity is measured by liquid-scintillation counting. NanthakumarN N, Dai D, Newburg D S, Walker W A. The role of indigenous microflorain the development of murine intestinal fucosyl- and sialyltransferases.FASEB J (Nov. 15, 2002) 10.1096/fj.02-0031fje (summary: FASEB J 2003;17:44-6).

FUT2 Genotyping Analysis

FUT2 genotyping can be performed by any standard method known in theart, for example SNP analysis or RT-PCR techniques. SNP's identified asleading to lower levels of FUT2 activity include TRP143TER (428G-A) andILE129PHE (385A-T). Complete deletion of the FUT2 gene is also observedin some non-secretors. Methods are well know in the art for determiningsingle nucleotide polymorphisms (SNPs), for example: Ahmadian A.,Gharizadeh B., Gustafsson A. C., Sterky F., Nyrén P., Uhlén M. andLundeberg J. Anal Biochem 280(1):103-10 (2000); Griffin T. J., Hall J.G., Prudent J. R. and Smith L. M. Proc Natl Acad Sci USA 96(11):6301-6(1999); Nickerson D. A., Kaiser R., Lappin S., Stewart J., Hood L. andLandegren U. Proc Natl Acad Sci USA 87(22):8923-7 (1990).

Other Assays

Ulex europaeus, a lectin that reacts specifically with alpha-1-fucose,can be used as the basis for detecting secretor antigens, and especiallyH-2. When ulex europaeus conjugated to colloidal gold is exposed to asample containing secretor antigens under suitable conditions, the ulexeuropaeus-colloidal gold will aggregate thereby producing detectablecolor change.

Briefly, ulex europaeus (UEA1) (Sigma-Aldrich; St Louis, Mo.) isconjugated to colloidal gold after determination of the minimum amountof UEA1 and optimal pH conditions required for stabilization of thecolloidal gold. On nitrocellulose strips, 2 μL of the untreated testsaliva is added and left to dry for 5 min; 200 μL of casein solution at1.0% in TBS are added and incubated for 3 min at room temperature. Thecasein solution is discarded; 150 μL of UEA1-colloidal gold conjugateare added at the optimal dilution in casein solution at 0.05% in TBS,and incubated for 10 min. A detectable color change indicates thepresence of secretor antigens in the sample. Control samples are salivasamples known to be H-2 positive or negative.

Methods of Treatment

The methods disclosed herein are also useful for the treatment of aninfant at risk for infectious and inflammatory disorders. Treatment cancompletely or partially abolish some or all of the signs and symptoms ofthe infectious or inflammatory disorder, decrease the severity ofsymptoms, delay their onset, or lessen the progression or severity ofsubsequently developed symptoms.

The methods of treatment are useful for infectious and inflammatorydisorders. The methods can be used for full-term infants, prematureinfants or extremely low birthweight infants. Full term infants includethose born between 37 and 42 weeks gestational age; premature infantsare typically those born at less than 37 weeks' gestational age.Extremely low birth weight (ELBW) is generally defined as a birth weightless than 1000 g (2 lb, 3 oz). The majority of ELBW infants are also theyoungest of premature newborns, usually born at 27 weeks' gestationalage or younger. Nearly 1 in 10 infants with low birth weight (<2500 g)are ELBW infants.

The infectious and inflammatory disorders that may be detected usingthemethods described herein include necrotizing enterocolitis (NEC),gastrointestinal infections, gastrointestinal inflammation, and sepsis.These disorders may be the result of disease, injury or of unknown causeand they may be influenced by one's genetic constitution.

In NEC, portions of the bowel undergo necrosis, i.e., tissue death.Although NEC affects the gastrointestinal tract it can, in severe cases,have profound systemic impact. Initial symptoms may be subtle and caninclude one or more of: feeding intolerance, delayed gastric emptying,abdominal distention and/or tenderness, ileus/decreased bowel sounds,and, in the advanced stages, abdominal wall erythema and hematocheziaSystemic signs are nonspecific and can include any combination of apnea,lethargy, decreased peripheral perfusion, shock (in advanced stages),cardiovascular collapse and bleeding diathesis (consumptioncoagulopathy). Nonspecific laboratory abnormalities can include thefollowing: hyponatremia, metabolic acidosis, thrombocytopenia,leukopenia or leukocytosis with left shift, neutropenia, prolongedprothrombin time, and activated partial thromboplastin time, decreasingfibrinogen, and rising fibrin split products (in cases of consumptioncoagulopathy). Although the exact etiology is not known, the etiologymay be multifactorial and involve any or all of abnormal bacterialflora, intestinal ischemia and/or reperfusion injury, and intestinalmucosal immaturity.

Gastointestinal infections in infancy include symptoms of diarrhea; thepresence of mucus or blood in stools; vomiting; dehydration; thirst;listlessness; dry mucous membranes; sunken fontanelles; decreased skinturgor; decreased capillary filling time; tachycardia; weak pulse,reduced blood pressure; and tenting or loss of skin turgor. Theinfectious agents can be bacterial, fungal, viral or parasitic. Examplesof bacterial agents include, but are not limited to, Staphylococcusspp., Staphylococcus aureus, Escherichia coli, Streptococcus spp.,Enterobacter spp., Klebsiella spp., Bacillus spp., Serratia spp.,Salmonella spp., Shigella spp., Campylobacter spp., Yersinia spp., andClostridium difficile. Examples of fungal agents include, but are notlimited to, Candida spp. Examples of parasitic organisms include, butare not limited to, Cryptosporidium spp., Giardia spp., Entamoebahistolytica, Cyclospora spp. Examples of viral organisms include, butare not limited to, rotavirus, cytomegalalovirus, enteric adenovirus,astrovirus, adenoviruses type 40 or 41, Norwalk and other noroviruses,and saporovirus.

The clinical signs of neonatal sepsis are nonspecific and are associatedwith characteristics of the causative organism and the body's responseto the invasion. Neonatal sepsis may be categorized as early or lateonset. Eighty-five percent of newborns with early-onset infectionpresent within 24 hours, 5% present at 24-48 hours, and a smallerpercentage of patients present between 48 hours and 6 days of life.Onset is most rapid in premature neonates. The infant manifests overtshock with pallor, poor capillary perfusion, and edema. These signs ofshock are indicative of severe compromise and are highly associated withmortality. Signs of sepsis can include any or all of cardiac signs,e.g., early stage pulmonary hypertension, decreased cardiac output,hypoxemia, progressive decreases in cardiac output with bradycardia andsystemic hypotension; metabolic signs, e.g., hypoglycemia,hyperglycemia, metabolic acidosis, and jaundice; neurologic signs, e.g.,meningitis, ventriculitis, arachnoiditis, vasculitis, phlebitis,thrombosis, cerebral edema, infarction, stupor and irritability,impairment of consciousness (i.e., stupor with or without irritability),coma, seizures, bulging anterior fontanel, extensor rigidity, focalcerebral signs, cranial nerve signs, nuchal rigidity, alterations incerebrospinal fluid (CSF), e.g., an elevated white blood cell count, anelevated protein level, a decreased CSF glucose concentration, andpositive culture results. Temperature instability is observed withneonatal sepsis and meningitis, either in response to pyrogens secretedby the bacterial organisms or from sympathetic nervous systeminstability. The neonate is most likely to be hypothermic. The infantmay also have decreased tone, lethargy, and poor feeding. Signs ofneurologic hyperactivity are more likely when late-onset meningitisoccurs. Other signs of neonatal sepsis include hematologic signs, e.g.,thrombocytopenia, abnormal white blood cell counts (WBC), abnormalneutrophil count (PMNs and immature forms), abnormal ratios ofimmature-to-total neutrophil count, disseminated intravascularcoagulation (DIC), abnormalities in prothrombin time (PT), partialthromboplastin time (PTT), and fibrinogen and D-dimer levels; andgastrointestinal signs, e.g., necrotizing enterocolitis.

Organisms that have been implicated in causing late-onset sepsis includecoagulase-negative Staphylococci, S. aureus, E. coli, Klebsiella spp.,Pseudomonas spp., Enterobacter spp., group B Streptococcus, Serratiaspp., Acinetobacter spp., and Candida spp. The infant's skin,respiratory tract, conjunctivae, gastrointestinal tract, and umbilicusmay become colonized from the environment, leading to the possibility oflate-onset sepsis from invasive microorganisms. Vectors for suchcolonization may include vascular or urinary catheters, other indwellinglines, or contact from caregivers with bacterial colonization.

Infants identified based on their glycan phenotype (secretor, Lewis, orsialyl antigen expression) as being at risk for infectious andinflammatory disorders, e.g., NEC, gastrointestinal infections, andsepsis, can be treated with therapies that include one or moreprotective agents. Unless the context indicates otherwise, we use theterm “agent” to broadly refer to any substance that affects a targetmolecule or a target region of the gastrointestinal system in aclinically beneficial way (e.g., to inhibit pathogens from binding tohost cell surface glycans). Useful protective agents include, forexample, human milk feeding, probiotic organisms, prebiotics, or α1,2fucosyl glycans.

The α1,2 fucosyl glycans are saccharides that include a fucose terminusin an α1,2 linkage and as such are homologues of secretor antigens,i.e., they include a minimal disaccharide precursor, or core sequence,covalently linked to a fucose residue in an α1,2 configuration. The coresequence can be either the lacto type I structure, galactose (β1-3)N-acetylglucosamine-R, which we abbreviate here as {Gal (β1-3)GlcNAc}-Ror the lacto type II structure galactose (β1-4) N-acetylglucosamine-R,which we abbreviate here as{Gal(β1-4)GlcNAc-R}, wherein R is an H, asmall radical, or another monosaccharide, disaccharide or polysaccharideor a glycoprotein or glycolipid. These saccharides can be freeoligosaccharides or conjugated and expressed as glycoproteins,glycolipids, or other structures. The conjugated and unconjugated formsof oligosaccharides are together classified as glycans. Thus, the α1,2fucosyl glycans can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,20, 24, 28, 32, 36 or more sugars; one or more of the sugars iscovalently linked to a fucose residue in an α1,2 configuration, so thatthe glycans can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 18 or more fucose residues. Examples of suitable α1,2 fucosylglycans include, without limitation, 2′-fucosyllactose (2′-FL);lacto-N-fucopentaose-I (LNF-I); lacto-N-difucohexaose I (LDFH I);lactodifucotetraose (LDFT), or such fucosylglycan epitopes attached to aglycolipid, glycopeptide, glycoprotein, mucin, or other scaffold, eitherin the soluble form or as part of a probiotic organism. The α1,2 fucosylglycans can be purified from natural sources, e.g., milk, milk productsor plant products, using method known to those in the art. Alternativelyor in addition, glycans can be synthesized chemically either fromnaturally occurring precursors or synthetic templates according tomethods known in the art. In addition, glycans can be synthesizedenzymatically, either in vitro, or in vivo using specifically engineeredmicroorganisms such as bacteria or yeasts, using biosynthetic enzymeswell known in the art

A protective agent can also be a probiotic organism, i.e., a livingmicroorganism that, when ingested by the host, can modify intestinalmicrobial populations in a way that benefits the host. Probioticorganisms may provide an increased barrier to translocation of bacteriaand bacterial products across mucosa, competitively exclude potentialpathogens, modify of host response to microbial products, and enhanceenteral nutrition in ways that inhibits the growth of pathogens such asKlebsiella pneumoniae, Escherichia coli, and Candida albicans.

Probiotic organisms generally include bacteria and yeast. The species ofprobiotic organism can vary, but suitable species for infants includeLactobacilli, e.g., Lactobacillus rhamnosus GG, L. acidophilus, L.casei, L. plantarum, L. reuteri; and Bifidobacteria, e.g.,Bifidobacterium infantis, B. bifidum, B. breve, B. animalis subsp.lactis, B. longum, as well as Streptococcus thermophilus. Useful yeastspecies include Saccharomyces boulardii and Kluyveromyces lactis.Probiotic organisms may be either naturally occurring or they may beengineered, i.e., organisms may be provided with genes that enable themto acquire desirable properties such as, but not limited to, the abilityto express secretor antigens. Probiotic organisms may be administeredseparately or in combination. Commercially available probioticformulations include, for example, Infloran® (Istituto SieroterapicoBerna, Como, Italy) which contains Lactobacillusacidophilus/Bifidobacterium infantis; ABC Dophilus (Solgar, Israel)which contains Bifidobacterium infantis, B. bifidum and Streptococcusthermophilus; and Dicoflor (Vitis Pharma, Warsaw, Poland) which containsL. rhamnosus GG. A protective agent can also be a prebiotic, i.e., anon-digestible food ingredient that beneficially affects the host byselectively stimulating the growth and/or the activity of one or alimited number of bacteria in the colon. In contrast to a probiotic,which introduces exogenous bacteria into the colonic microbiota, aprebiotic stimulates the growth of one or a limited number of thepotentially health-promoting indigenous microorganisms e.g.,Bifidobacteria or Lactobacteria. Examples of prebiotics includefructo-oligosaccharides, e.g., inulin, xylooligosaccharides andgalacto-oligosaccharides. Prebiotics can be isolated from naturalsources e.g., chicory roots, soybeans, Jerusalem artichokes, beans,onions, garlic, oats, wheat and barley.

One useful prebiotic is inulin, a type of fructan (polymer of fructose).Inulin-type fructans are composed of β-D-fructofuranoses attached byβ-2,1 linkages. The first monomer of the chain is either aβ-D-glucopyranosyl or β-D-fructopyranosyl residue. Various forms ofinulin and inulin fragments are available from commercial sources, e.g.,inulin with a degree of polymerization (DP) from 2 to 60 is extractedfrom chicory roots (Raftiline; Orafti, Tienen, Belgium); oligofructose,which is produced by partial enzymatic hydrolysis of inulin, has a DP<10(Raftilose; Orafti) and the inulin from which the small-molecular-weightoligomers have been eliminated is called high-performance inulin(Raftiline H P; Orafti). With the use of sucrose as a substrate and a1,2-β fructan in a fructosyltransferase-catalyzed reaction, a syntheticlow-molecular-weight fructan is produced that has a DP<4 (Neosugar orActilight; Beghin-Meji Industries, Paris).

Protective agents may be administered directly to a patient, eithersingly or in combination. Generally, the protective agents can besuspended in a pharmaceutically acceptable carrier (e.g., physiologicalsaline or a buffered saline solution) to facilitate their delivery.Encapsulation of the protective agents in a suitable delivery vehicle(e.g., polymeric microparticles or implantable devices) may increase theefficiency of delivery. A composition can be made by combining any ofthe protective agents provided herein with a pharmaceutically acceptablecarrier. Such carriers can include, without limitation, sterile aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents include mineral oil, propylene glycol, polyethyleneglycol, vegetable oils, and injectable organic esters. Aqueous carriersinclude, without limitation, water, alcohol, saline, and bufferedsolutions. Preservatives, flavorings, and other additives such as, forexample, antimicrobials, anti-oxidants (e.g., propyl gallate), chelatingagents, inert gases, and the like may also be present. It will beappreciated that any material described herein that is to beadministered to a mammal can contain one or more pharmaceuticallyacceptable carriers.

Alternatively, or in addition, protective agents may be combined with aninfant's food source, e.g., expressed breast milk or commerciallyavailable infant formula. Any composition described herein can beadministered to any part of the host's body for subsequent delivery tothe gastrointestinal tract. A composition can be delivered to, forexample, the mouth, nasal mucosa, blood, lungs, intestines, muscletissues, skin, or the peritoneal cavity of a mammal. In terms of routesof delivery, a composition can be administered by intravenous,intracranial, intraperitoneal, intramuscular, subcutaneous,intramuscular, intrarectal, intravaginal, intratracheal, intradermal, ortransdermal injection, by oral or nasal administration, or by gradualperfusion over time. In a further example, an aerosol preparation of acomposition can be given to a host by inhalation.

The dosage of protective agent that is required will depend on thenature of the agent, route of administration, the nature of theformulation, the nature of the patient's illness, the patient's size,weight, surface area, age, and sex, other drugs being administered, andthe judgment of the attending clinician. Wide variations in the neededdosage are to be expected in view of the variety of protective agentsand the differing efficiencies of various routes of administration.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-,10-times, or more). Encapsulation of the protective agents in a suitabledelivery vehicle (e.g., polymeric microparticles) may increase theefficiency of delivery.

The duration of treatment with any composition provided herein can beany length of time from as short as one day to as long as the increasedrisk might be suspected clinically, for example, through the period ofneonatal intensive care unit stay or through infancy. For example, aprotective agent can be administered several times per day, once a day,or once a week (for, for example, 4 weeks to several months). It is alsonoted that the frequency of treatment can be variable. For example, theprotective agents can be administered once (or twice, three times, etc.)daily, weekly, or monthly.

An effective amount of any composition provided herein can beadministered to an individual at risk of disease or in need oftreatment. The term “effective” as used herein refers to any amount thatinduces a desired response while not inducing significant toxicity inthe patient. Such an amount can be determined by assessing a patient'sresponse after administration of a known amount of a particularcomposition. In addition, the level of toxicity, if any, can bedetermined by assessing a patient's clinical symptoms before and afteradministering a known amount of a particular composition. It is notedthat the effective amount of a particular composition administered to apatient can be adjusted according to a desired outcome as well as thepatient's response and level of toxicity. Significant toxicity can varyfor each particular patient and depends on multiple factors including,without limitation, the patient's disease state, age, and tolerance toside effects.

The protective agents provided herein can be administered in conjunctionwith other prophylactic or therapeutic modalities to an individual atrisk for an infectious or inflammatory disorder, e.g., NEC, agastrointestinal infection or sepsis. The protective agents can be givenprior to, simultaneously with or after treatment with other agents orregimes. Other treatments can include administration of antibiotics, forexample, vancomycin, kanamycin, gentamicin, cefotaxime, clindamycin ormetronidazole, enteral administration of IgG and IgA together, aminoacid supplementation, the use of platelet-activating factor (PAF)antagonists or PAF-acetylhydrolase administration, polyunsaturated fattyacid administration, epidermal growth factor administration, andantenatal corticosteroids. The protective agents can also beadministered along with or in addition to, other feeding regimes,including judicious administration of human milk feeding, infantformula, parenteral fluids, delayed or slow feeding.

Also provided are methods of determining the course of treatment for aninfant who has been identified, based on secretor status, as being atrisk for NEC or a gastrointestinal infection. The levels of secretorantigens, e.g., α1,2 fucosyl glycans, in the infant's food source can becompared with levels of the same secretor antigens in a referencesample; the levels of secretor antigens in the food source can beclassified as reduced or elevated relative to those secretor antigens ina reference sample. Those infants whose secretor status indicates thatthey are at risk for NEC and gastrointestinal infections and whose foodsource also contains reduced levels of secretor antigens can be treatedwith one or more of α1,2 fucosyl glycans, probiotic organisms orprebiotics.

The infant's food source can be breast milk, either from the infant'sown mother or a donor source, or a commercial infant formula. The levelof one or more secretor antigens can be assayed in human milk andformula e.g., by ELISA, chromatography or another method and compared tothose in a reference sample as described above.

Human milk oligosaccharides typically contain a lactose moiety at thereducing end and a fucose at the nonreducing end. The addition of fucoseto an oligosaccharide by an α1,2 linkage is catalyzed primarily by thefucosyltransferase produced by the secretor gene, Se (FUT2); theaddition of fucose by an α1,3 or α1,4 linkage is catalyzed byfucosyltransferases produced by the Lewis gene, Le (FUT3) or other α1,3transferase genes (FUT4, 5, 6, 7, and 9) of this family. Variation inthe activities of the 2- and 3/4-fucosyltransferases can result frominactive or partially active genetic polymorphisms. Such variation canproduce milk phenotypes that vary in relative quantities of specificfucosyloligosaccharides. Women who are nonsecretors do not expressmeasurable 2-linked fucosyloligosaccharides in their milk or otherbodily fluids. However, the expression of milk fucosyloligosaccharidescan vary even among secretors and the ratio of 1,2-linkedfucosyloligosaccharides to those that contain only 1,3- and 1,4-linkedfucose declines exponentially over the first year of lactation.

Examples of suitable α1,2 fucosyl glycans include, without limitation,2′-fucosyllactose (2′-FL), which is homologous to H-2;lacto-N-fucopentaose-I (LNF-I), which is homologous to H-1;lacto-N-difucohexaose I (LDFH I), which is homologous to Lewis^(b);lactodifucotetraose (LDFT), which is homologous to Lewis^(y), as well aslacto-N-difucohexaose II (LNF-II), which is homologous to Lewis^(a); andlacto-N-difucohexaose III (LNF-III), which is homologous to Lewis'. Thecore type 1 structure, lacto-N-tetraose (LNT), is a terminalGalβ1,3GlcNAc on lactose. Lactose is the core for the most abundant type2 structures in milk (2′-FL, 3-FL, and LDFT), whereaslacto-N-neotetraose, Galβ1,4GlcNAc on a lactose terminus, is the corefor LNF-III. In other tissues, Lewis structural moieties are based on alactosamine backbone (Gal-GlcNAc); however, the most prevalent type 2fucosyloligosaccharides in human milk are synthesized from lactose(Gal-Glc) and therefore are defined as the glucose analogs to the type 2Lewis structures. The epitopes of these structures can also be foundexpressed in various glycoconjugates of milk, and these glycoconjugatesmay be used for prophylactic and/or therapeutic purposes.

Infants whose food supply contains a reduced level of secretor antigensmay be supplemented enterally by feeding α1,2 fucosyl glycans, probioticorganisms or prebiotics as described above.

EXAMPLES Example 1 Bacterial Colonization Induces Change in IntestinalGlycoylation

Expression of fucosylated and sialylated glycans in gut change duringdevelopment, reminiscent of the changes seen in milk over the course oflactation. In the mouse small intestine, Fut2 mRNA andα1,2/3-fucosyltransferase activity increase abruptly at weaning, whileexpression of sialyltransferase activity decreases. The inversion ofthese two enzyme activities coincides with an abrupt change of mucosalglycan expression at weaning and a change in composition of gutmicrobiota. Whether induction of these changes was through an intrinsicgenetic program, or by exogenous control by diet or adult microflora,was tested as described below.

The upregulation of fucosyltransferase that normally would occur atweaning does not occur in germ-free mice. However, whenever post-weaninggerm-free mice are colonized, fucosyltransferase and fucosylation areinduced. This suggested that bacterial colonization was inducingfucosylation of the mucosal surface. This was confirmed in mature micethat were depleted of bacteria (BD) by drinking a mixture ofantibiotics. After two weeks, Fut2 mRNA and fucosyltransferase activitydropped to the levels seen in germ-free mice, and fucosylglycans (Ulexeuropaeus agglutinin 1 [UEA-1] staining) were no longer expressed in thecolon. Cessation of antibiotic treatment and repletion with normalmicrobiota (XBD) caused a recovery of Fut2 mRNA and fucosyltransferaseactivity to levels of normally colonized mature gut, and full expressionof fucosylglycans on the mucosal surface. This confirmed thatfucosylation of the gut is controlled by its colonization.

It was hypothesized that the mechanism whereby extracellularcolonization results in the intracellular activation of the ERK and JNKpathway involves undefined transmembrane receptors. A family oftransmembrane receptors, the toll-like receptors (TLR), were alreadyknown to sense the extracellular presence of pattern recognitionmolecules unique to microbes and transmit signals to the nucleus throughtranscellular signal transduction pathways. Accordingly, we testedwhether one of the TLR family members might be responsible for thecommunication between the bacteria and the gut mucosa that results inbacterial-induced Fut2 mRNA and fucosyltransferase activity in thecolonic mucosa. Like wild-type mice, mice with mutations in TLR2 areable to express normal fucosylation that is lost with a loss ofcolonization (FIG. 12, next page). In contrast, mutants of TLR4 or itsdownstream mediator, MyD88, do not express the full level offucosylation, and this fucosylation is not affected by loss ofcolonization. This is consistent with the TLR4 signaling pathway beingnecessary for bacteria-induced mucosal fucosylation. To test whetheractivation of TLR4 per se in bacteria-depleted mice is sufficient toactivate the transduction pathways that stimulate Fut2 expression, thespecific ligand for TLR4, LPS, was administered in drinking water tobacteria-depleted mice. Fucosyltransferase activity and Fut2 mRNA of theBD mouse colon recovers to normal adult levels when treated withultra-pure LPS. In contrast, in BD mice treated with peptidoglycan (PG),the ligand for TLR2, the level of fucosyltransferase activity and Fut2mRNA remains at the lower levels of BD mice. These data strongly suggestthat binding and stimulation of TLR4 in bacterially depleted mice is thecritical signal that is both necessary and sufficient for adult gutmicrobiota to signal the epithelial nuclear events that result infucosylation of the gut.

If binding to fucosylated TLR4 is the essential signal for thecolonization-induced mucosal fucosylation, the subset of the microbiotathat bind to fucose would be expected to recapitulate this phenomenon.This hypothesis was tested with a fucose-utilizing species of the mixedmicrobiota. Bacteroides fragilis is a specific fucose-utilizingbacterium found in typical mature mammalian microbiota. Monocolonizationof bacterially depleted mice with B. fragilis induced fucosylation tothe same extent as recolonization with mixed microbiota, mediatedthrough induced Fut2 mRNA, consistent with the fucose-utilizing bacteriaof the mixed microbiota being responsible for the induction of fucoseexpression. If this were so, a mutant B. fragilis made incapable ofutilizing fucose would be expected to lose the ability to signal theinduction of fucosylation in the mucosa, as observed in the right panel.Thus, binding to one set of fucosylated epitopes, seemingly fucosylatedTLR4, in bacterially depleted mice by fucose-utilizing bacteria seems tobe sufficient for the induction of the fucosylated phenotype on theintestinal mucosa.

Example 2 Histo-blood Group Antigens (Glycans) in Saliva of HospitalizedInfants

Thirty-six infants hospitalized in Cincinnati area Newborn IntensiveCare Units between 24 and 42 weeks gestational age (GA) were enrolledbetween May and December 2005. Twelve subjects per group were stratifiedby gestational age into three groups, 24 to 28 weeks, 29 to 32 weeks,and greater than or equal to 33 weeks gestational age at birth. Infantsdiagnosed with major congenital anomalies were excluded. InstitutionalReview Boards at Cincinnati Children's Hospital Medical Center, GoodSamaritan Hospital, and University Hospital approved the study. Informedwritten consent was obtained from the parents. Maternal demographicinformation collected included maternal age, race, obstetric history,complications encountered in the current pregnancy, and maternalmedications during pregnancy. Clinical and demographic data recorded atenrollment of subjects included race, gender, gestational age, birthweight, length and head circumference. Clinical data recorded on studyinfants during their hospital course included requirement and durationof respiratory support, initiation and type (human milk or formula) ofenteral nutrition, episodes of culture-proven sepsis, occurrence ofnecrotizing enterocolitis (Bell's stage 2 or greater), and history ofantibiotic use.

Specimen Collection.

Saliva specimens were obtained at enrollment and every two weeks whilesubjects remained hospitalized. A maximum of five samples were collectedfrom each subject. Saliva specimens were collected one to two hoursafter feeding by clearing the mouth of residual milk or formula withsoft gauze and inserting a sterile cotton swab. Once visibly saturatedwith saliva, the cotton swab was transferred into a specimen container.Specimens were held briefly at 4° C. then transferred to −80° C. A totalof 107 saliva samples were collected. Saliva-saturated cotton swabs wereallowed to thaw in 1 ml phosphate buffered saline (PBS) for 5 minutes.The specimens were then centrifuged at 10,000×g for 10 minutes andsupernatants were collected. Each specimen was boiled at 100° C. for 10minutes and placed at 4° C. overnight. Samples were again centrifuged at10,000 g× for 10 minutes, the supernatants were collected, separatedinto 100 ml aliquots and placed at −80° C.

Optimal dilutions of saliva samples were determined for each antigendetection assay. Saliva specimens diluted 1:50 were used for detectionof Le^(a), Le^(x), H-1, H-2, sialyl Le^(a) and sialyl Le^(x) antigens.Saliva specimen diluted 1:125 were used for Le^(y), whereas dilutions of1:250 were used for Le^(b) antigen detection. For quantitative analysisof saliva samples from an individual subject, a 1:50 dilution was usedfor all antigens. Samples were coated onto microtiter plates (DynexImmunlon) overnight at 4° C. After blocking with 5% Blotto, monoclonalantibodies (MAbs) specific to Lewis and ABH antigens were used at adilution of 1:100. The following MAbs specific to human histo-bloodgroup antigen types were used for histo-blood group phenotypedeterminations. MAbs BG-4 anti-H type 1, BG-5 anti-Le^(a), BG-6anti-Le^(b), BG-7 anti-Le^(x), and BG-8 anti-Le^(y) were purchased fromSignet Laboratories. MAb BCR9031 anti-H type 2, BCR9010 anti-A, and BCRM11007 anti-B were purchased from Accurate Chemical and ScientificCorporation. MAbs for Sialyl Le^(a) and Le^(x) were products of EMD,catalog number 565942 and 565953 respectively. After incubation for 1hour at 37° C., horseradish peroxidase (HRP) conjugated goat anti-mouseIgG, IgG3 or IgM antibodies were added. After each step, the plates werewashed five times with PBS/Tween solution. Colorimetric reactions weredetected using a TMB kit (Kirkegard & Perry Laboratories), and read at awavelength of 450 nm using an EIA spectrum reader (Tecan).

Shown in Table 1 is the average optical density values (±the standarderror) obtained by ELISA in study week 1 and prior to discharge for allinfants ever found to have a detectable level of that antigen in asaliva sample. Each antigen was considered separately. This analysisindicated that most secretor antigens (Lewis^(b), Lewis^(y), and H-2)were present in premature infants from birth, and that secretor antigenexpression increased postpartum. As shown in Table 1, levels of secretorantigens (shown in boldface type) were higher in the samples collectedat subsequent time points, i.e., those samples taken from infants priorto discharge. Levels of Lewis^(b), Lewis^(y) and Lewis^(b) and Lewis^(y)showed a statistically significant increase over time; levels of theother secretor antigens, H-1 and H-2, also increased, although the rateof increase did not approach statistical significance.

TABLE 1 Glycan Expression in Saliva of Premature Infants PostpartumPostpartum Week 1 Pre-Discharge change Antigen N Saliva Sample* SalivaSample* p-value** Secretor H-1 20 0.18 (0.08) 0.29 (0.06) 0.13 H-2 301.45 (0.20) 1.80 (0.21) 0.41 Lewis^(b) 28 1.92 (0.20) 4.02 (0.45) 0.0005Lewis^(y) 31 2.78 (0.24) 3.90 (0.29) 0.024 Lewis (only) Lewis^(a) 321.22 (0.46) 1.27 (0.54) 0.82 Lewis^(x) 26 0.71 (0.19) 0.95 (0.32) 0.40Sialyl SLe^(a) 36 3.63 (0.61) 3.10 (0.89) 0.29 SLe^(x) 35 1.85 (0.27)1.61 (0.36) 0.49 *Values are expressed as the mean optical density; thestandard error is in parentheses. **p-values were determined using aStudent's t-test.

Example 3 Antibiotic Use and Glycan Expression Phenotype

The relationship between secretor antigen expression, postpartum age,gestational age and antibiotic use was analyzed using a GeneralizedEstimating Equation (GEE). Saliva samples were collected from 24 infantsin the first week of age postpartum and “prior to discharge”. Levels ofhisto-blood group antigens Lewis^(a), Lewis^(x), Lewis^(b), Lewis^(y),H-1, H-2, Sialyl-Lewis^(a) (SLe^(a)) and Sialyl-Lewis^(x) (SLe^(x)) weremeasured by ELISA according to the method described in Example 1.Infants were classified according to gestational age at birth intogroups of 22-28 weeks, 29-34 weeks, and 35-40 weeks. For GEE analysis,histo-blood group antigen O.D. values were used as dependent variables(designated as “Model” in column 1 of Table 2) and week of agepostpartum, gestational age group, and number of days of antibiotic usewere included as independent variables. The resulting beta coefficientsfor the GEE analysis are shown in Table 2. The GEE analysis indicatedthat the courses (defined as antibiotic treatment of the infant from 1-5days) was associated with significantly decreased expression of secretorantigens postpartum.

TABLE 2 Beta coefficients (SE) from Generalized Estimating Equation(GEE) analysis: Histoblood group antigen O.D. values (dependentvariable) measured in samples from 24 infants with two saliva samplescollected at Week 1 and prior to hospital discharge (3-7 weeks afterbirth). Week of age postpartum, gestational age group, and number ofdays of antibiotic use were included as independent variables Week ofage Gestational Age No. of courses of Model postpartum Group* antibioticuse Lewis b and y   0.54 (.17)*** 0.005 (.39) −0.33 (.37) (combined O.D.values) Sialyl Le X −0.10 (.08) −0.17 (0.21)   0.52 (.14)****Gestational age groups: 1 = 24-28 wks, 2 = 29-32 wks; 3 = 33-40 wks atbirth, ***p ≦ 0.001

Example 4 Extremely Low Birthweight Infant (ELBW) Outcomes Study

The relationship between secretor antigen expression, clinical outcomeand necrotizing enterocolitis (NEC) was analyzed in a cohort of 192extremely low birthweight (<1000 gram) infants seen in Cincinnatineonatal intensive care units, with saliva samples collected in week 1(days 1-7) and week 2 (days 8-14) after birth. This was a prospectivestudy conducted at three hospitals that provide level III neonatalintensive care within the Cincinnati region, with infants enrolledbetween 2002-2004. Exclusion criteria included the presence of majorchromosomal or congenital anomalies, diagnosis of cystic fibrosis, or amedical condition judged by the attending neonatologist to beincompatible with survival beyond the first week of life. Afterenrollment, a saliva sample was obtained along with demographic andclinical data. Infants were followed with once-weekly saliva samplecollections. Saliva was collected with sterile cotton-tipped swabsplaced in the mouth of the infant and saturated with saliva, by nursingor research staff between 5:00 am and 10:00 am before feeding. Sampleswere frozen at −80 C. Saliva was extracted from the swab by removing thecotton portion of the swab, placing it in a 1 mL syringe, and elutingthe contents with 250 mL of normal saline. FIG. 6 is a scatter plot ofthe results.

The rate of NEC in the study population was 7.8% (n=15); the rate oflate onset sepsis was 34.9% (n=67); the rate of death was 9.3% (n=18).Of the 15 NEC cases, there was a 60% case fatality and the rate of deathdue to NEC among the 192 infants studied was 4.7% (n=9).

Levels of histo-blood group antigens Lewis^(a), Lewis_(x), Lewis^(b),Lewis^(y), H1, H2, Sialyl-Lewis^(a) (SLe^(a)) and Sialyl-Lewis^(x)(SLe^(x)) were measured by ELISA according to the method described inExample 1 in a single banked saliva sample taken from each infant.Individuals expressing one or more antigens containing an α1,2-linkedfucose were designated as secretors. Twenty percent (20%), i.e., 39infants, were classified as non-secretors (no detectable secretorantigen); 153 infants (80%) were classified as secretors.

Classification and Regression Tree (CART) analysis was used to identifythe high-risk subgroups for NEC or death using the glycan (secretor,Lewis, and sialyl antigen) values of each individual. CART analysis isan established statistical method that uses tree-based partitioning toidentify algorithms for diagnostic or prognostic markers of risk inclinical studies such as this. An empirical statistical technique basedon recursive partitioning analysis, the method does not requireparametric assumptions, and involves the segregation of different valuesof continuous or categorical data through a decision tree composed ofprogressive binary splits. Every value of each predictor variable isconsidered a potential split, and the optimal split is selected based onminimizing misclassification of cases and non-cases using an “impuritycriterion”, which is the reduction in the residual sum of squares thatwould occur with a binary split of the data at that node. All sevenantigens were analyzed, one at a time, using CART to generate theoptimal cut-point for each variable. The categorical variables createdfrom that step (partially shown in FIG. 1) were then re-entered into asecond CART model. In this step, H-2 emerged as the first split (seeFIG. 2), defining 73 infants into a high risk group at the lowest38^(th) percentile of values and below (O.D. value <0.627). This grouphad 18 cases of NEC or death (incidence 24.7%) compared with 6 cases ofNEC or death among the 119 infants classified into the H-2 low-riskgroup (incidence 5.0%, P<0.0001). Among the 73 infants classified intothe high-risk group by their salivary H-2 (the “low-secretors” andnon-secretors), a second split occurred in the CART model: 19 wereidentified as low-risk (1 case of NEC or death, incidence 5.2%) and 54were identified as high risk (17 cases of NEC or death, incidence31.5%). This model identified one high-risk group (the low- andnon-secretors, 31.5% risk) and two low-risk groups (the 119 who werehigh secretors and the 19 who were non- and low-secretors but also lowsLe^(a)) with nearly identical risk approaching 5%. Lastly, these 3nodes were re-entered into the final CART model: the result was a binarysplit identifying the high-risk group vs. all others (FIGS. 2 and 3).This single split, combining H-2 and sLe^(a) predictive high-riskcut-points, was found by receiver operating curve area under the curveanalysis to have an overall predictive value of 77. The CART analysis issummarized in FIGS. 5 and 6.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

What is claimed: 1.-47. (canceled)
 48. A method for treating agastrointestinal infectious or gastrointestinal inflammatory disorder inan individual, the method comprising: administering to the individual aneffective amount of a composition comprising one or more alpha-1,2fucosyl glycans.
 49. The method of claim 1, wherein the one or morealpha-1,2 fucosyl glycans each include a fucose terminus in anα1,2-linkage to a core sequence, which is Gal(β1-3)GlcNAc-R₁ orGal(β1-3)GlcNAc-R₂, each of R₁ and R₂ independently being H, a smallradical, a monosaccharide, a disaccharide, or a polysaccharide.
 50. Themethod of claim 2, wherein the one or more alpha-1,2 fucosyl glycans areselected from the group consisting of 2′-fucosyllactose (2′-FL),lacto-N-fucopentaose-I (LNF-I), lacto-N-difucohexaose I (LDFH I), andlactodifucotetraose (LDFT).
 51. The method of claim 1, wherein the oneor more alpha-1,2 fucosyl glycans are conjugated to a scaffold.
 52. Themethod of claim 4, wherein the scaffold is a glycoprotein, aglycopeptide, a glycolipid, or a mucin.
 53. The method of claim 1,wherein the composition further comprises one or more probioticorganisms.
 54. The method of claim 6, wherein the one or more probioticorganisms are selected from the group consisting of Lactobacillirhamnosus GG, Lactobacilli acidophilus, Lactobacilli casiei,Lactobacilli plantarum, Lactobacilli reuteri, Bifidobacterium infantis,Bifidobacterium bifidum, Bifidobacterium Breve, Bifidobacterium animalislactis, Bifidobacterium longum, Streptococcus thermophiles,Saccharomyces boulardii, and Kluyveromyces lactis.
 55. The method ofclaim 1, wherein the composition further comprises one or moreprebiotics.
 56. The method of claim 8, wherein the one or moreprebiotics are fructo-oligosaccharides.
 57. The method of claim 9,wherein the fructo-oligosaccharides are inulin, xylooligosaccharides, orgalactooligosaccharides.
 58. The method of claim 1, wherein theindividual is an infant.
 59. The method of claim 1, wherein theinfectious or inflammatory disorder is necrotizing enterocolitis,gastrointestinal infection, gastrointestinal inflammation, or sepsis.60. The method of claim 11, wherein the infectious or inflammatorydisorder is necrotizing enterocolitis, gastrointestinal infection,gastrointestinal inflammation, or sepsis.
 61. The method of claim 1,wherein the composition further comprises a pharmaceutically acceptablecarrier.